In presently used centralized testing systems samples are taken and shipped to a central lab for analysis, e.g. in clinical diagnostics, life sciences, biodefense, food and water industries, and agricultural and environmental sensing. This leads to significant costs and long periods of time to result. In contrast, portable sample preparation devices can introduce a shift not only in the instrumentation but in the entire measurement procedure by performing measurements anytime and anywhere. Sample preparation is the limiting element for such devices, since this process is typically done manually, but it also has been automated by mechanizing manual processing methods through robotic systems. Commercially available instruments are thus large, expensive and complex with significant reagent and consumable usage.
Microfluidics offers reduced reagent use, increased specificity and robustness, integration and automation, potential for parallel analysis, cost-effective devices fabricated by injection molding, and controlled channel designs. Lab-on-a-chip devices are thus particularly interesting for portable sample preparation modules as well as for cost-effective and automated lab instruments.
For the transport and separation of charged molecules in capillaries and micro-channels, electrophoresis is often employed and has found widespread applications. However, the sensitivity and selectivity to accurately handle and separate substances in tubings is limited, thus requiring efficient sample preconcentration methods.
One approach to preconcentrate a sample is isotachophoresis (ITP), which allows simultaneous separation of several analytes. ITP uses an imposed electrophoretic mobility gradient to create concentrated analyte zones with nondispersing interfaces in an elongated channel. Analyte ions to be stacked and separated are typically introduced between a leading (LE) and a trailing electrolyte (TE) with an effective mobility respectively higher and lower than those of the analytes. Under the influence of an electric field, analyte ions redistribute themselves into sequential zones in order of reducing effective mobility (starting from LE to TE). After initial transients, ITP based separations typically result in adjacent, contiguous zones of analytes moving at identical speed downstream in the main separation channel.
For the separation of charged components, U.S. Pat. No. 6,685,813 of Williams is based on the titration of analytes. The method involves loading a microchannel with a sample, placed between a TE having a selected concentration of a titratable species, and an LE. When a voltage is applied over the microchannel, charged components stack by ITP, and electrolytic hydroxyl or hydrogen ions migrate into the TE, titrating the species therein which then overtake the charged sample components and thus separate by zone electrophoresis.
There are several patent applications on ITP based preconcentration of analytes combined with their separation by electrophoresis and analysis. US Patent Application Publication No. 2006/0254915 of Hirokawa describes a microchip electrophoresis method for sample preconcentration and separation in two individual steps. Sample concentration is performed by ITP, followed by separation using zone electrophoresis or gel electrophoresis. Similarly, US Patent Application Publication No. 2002/0189946 of Wainright and Williams is drawn to a simple two-electrode injection scheme with isotachophoretic stacking, followed by zone electrophoretic separation in the same channel. Further, US Patent Application Publication No. 2005/0133370 of Park et al. discloses methods and devices for spatially separating at least first and second components by means of a spacer, which components are stacked by ITP and injected in different channel segments for their separation by mobility therein.
More specifically, U.S. Pat. No. 5,817,225 by Hinton describes an electrophoretic unit for the purification, concentration and size fractionation of nucleic acids contaminated by organic acids using specific LE and TE chemistries. International patent application WO 2009/079028 A1 of Young also employs gels for the concentration of proteins and DNA using ITP. The method is drawn to simultaneously co-purify and concentrate nucleic acids and protein targets with positive and negative net charge into a single volume, which are initially placed in the middle of a gel such that ITP will run in two directions towards both a positive and negative electrode when a voltage is applied.
A method to directly detect analytes that are per se undetectable using directly detectable spacer compounds has been disclosed by Santiago under US Patent Application Publication No. 2008/0197019 A1. These charged molecules are concentrated and separated into zones using ITP, and a displacement between the zones of directly detectable spacer compounds is used to determine the presence of the analyte that is not directly detectable.
Two international patent applications disclose partially using ITP for the concentration and separation of molecules within their systems. WO 2008/082876 A1 of Balgley provides a method for performing off-line multi-dimensional separation and analysis of a sample, including the separation of the heterogeneous biomolecular sample into a plurality of fractions using a partial capillary ITP mechanism. WO 2008/053047 A2 of Weber focuses on a specific type of free-flow electrophoresis (FFE), free-flow ITP (FFITP), performed in an almost quadratic electrophoresis chamber to separate different analytes.
However, kits for the ITP based extraction of specific fractions from a complex sample using custom electrolytes and disposable modules, which can be integrated in portable or handheld devices or automated for cost-effective lab instruments have not been disclosed so far.